The current diphtheria epidemic in the former Soviet Union has resulted in a heightened interest in the bacterium Corynebacterium diphtheriae, the causative agent diphtheria. With the exception of diphtheria toxin, no studies have been done to identify virulence determinants in this important bacterial pathogen. Additionally, little is known on how C. diphtheriae invades, colonizes and survives within the human host. The acquisition of iron is an important virulence determinant for many bacterial pathogens, and numerous virulence factors including diphtheria toxin are regulated by iron. In the current study, C. diphtheriae was examined for the capability to utilize various host compounds as iron sources. C. diphtheriae C&(-) acquired iron from heme, hemoglobin, and transferrin. A siderophore uptake mutant of strain C7 was unable to utilize transferrin, but was unaffected in acquisition of iron from heme and hemoglobin which suggests that C. diphtheriae possesses a novel mechanism for utilizing heme and hemoglobin as iron sources. Mutants of C. diphtheriae and C. ulcerans that are defective in acquiring iron from heme and hemoglobin were isolated. A clone obtained from a C&(-) genomic plasmid library complemented several of the C. ulcernas and C. diphtheriae mutants. The nucleotide sequence of the gene (humO) required for complementation was determined, and it was revealed that the predicted product, HmuO, has 33% identity and 70% similarity with the human heme oxygenase enzyme HO-1. It is proposed that the HmuO protein is essential for the utilizatino of heme as an iron source by C. diphtheriae and that the heme oxygenase activity of HmuO is involved in the release of iron from heme. This is the first report of a bacterial gene that has homology to heme oxygenases.